Manipulation of dominant male sterility

ABSTRACT

Compositions and methods for modulating male fertility in a plant are provided. Compositions comprise nucleotide sequences, and fragments and variants thereof, which modulate male fertility. Further provided are expression cassettes comprising the male fertility polynucleotides, or fragments or variants thereof, operably linked to a promoter, wherein expression of the polynucleotides modulates the male fertility of a plant. Various methods are provided wherein the level and/or activity of the sequences that influence male fertility is modulated in a plant or plant part. In certain embodiments, the plant is polyploid.

CROSS REFERENCE

This application is a continuation of U.S. Non Provisional applicationSer. No. 14/203,698 filed Mar. 11, 2014 which claims the benefit of U.S.Provisional Application No. 61/778,069, filed Mar. 12, 2013, and U.S.Provisional Application No. 61/788,950, filed Mar. 15, 2013, both ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to influencing male fertility.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant or a genetically identical plant. A plant is cross-pollinated ifthe pollen comes from a flower on a genetically different plant.

In certain species, such as Brassica campestris, the plant is normallyself-sterile and can only be cross-pollinated. In self-pollinatingspecies, such as soybeans and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower.

Bread wheat (Triticum aestivum) is a hexaploid plant having three pairsof homologous chromosomes defining genomes A, B and D. The endosperm ofwheat grain comprises 2 haploid complements from a maternal cell and 1from a paternal cell. The embryo of wheat grain comprises one haploidcomplement from each of the maternal and paternal cells. Hexaploidy hasbeen considered a significant obstacle in researching and developinguseful variants of wheat. In fact, very little is known regarding howhomologous genes of wheat interact, how their expression is regulated,and how the different proteins produced by homologous genes functionseparately or in concert.

An essential aspect of much of the work underway with genetic malesterility systems is the identification of genes influencing malefertility and promoters associated with such genes. Such genes andpromoters can be used in a variety of systems to control male fertilityincluding those described herein.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for modulating male fertility in a plant areprovided. Compositions comprise nucleotide sequences, and fragments andvariants thereof, which modulate male fertility. Further provided areexpression cassettes comprising one or more polynucleotides, operablylinked to a promoter, wherein expression of one or more polynucleotidesmodulates the male fertility of a plant. Various methods are providedwherein the level and/or activity of a polynucleotide that influencesmale fertility is modulated in a plant or plant part.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Alignment of the wheat MS45 promoter regions of the A,B, and D genomes (SEQ ID NOs: 1, 2, and 3, respectively). A consensussequence is also provided (SEQ ID NO: 16).

FIG. 2. Alignment of wheat MS45 promoter consensus (SEQ ID NO: 4) withZmMS45 promoter region (SEQ ID NO: 5). A consensus sequence is alsoprovided (SEQ ID NO: 14).

FIG. 3. Alignment of wheat MS45 promoter consensus (SEQ ID NO: 4) withwheat promoter inverted repeat (pIR) sequence (SEQ ID NO: 6). Aconsensus sequence is also provided (SEQ ID NO: 15).

FIG. 4. Restoration of fertility by Gain of Function: GOF-MF.

FIG. 5. Restoration of fertility by Gain of Function: GOF-MF; MS45.

FIG. 6. Restoration of fertility by Gain of Function: GOF-pIRMSp.

FIG. 7. Restoration of fertility by Gain of Function: GOF-pIRMSp;5126:DAM.

DETAILED DESCRIPTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Male Fertility Polynucleotides

Compositions disclosed herein include polynucleotides and polypeptidesthat influence male fertility. In particular, wheat MS45 promotersequences are provided comprising nucleotide sequences set forth in SEQID NO: 1, 2 or 3, or fragments or variants thereof, such as SEQ ID NO: 4or 6.

Sexually reproducing plants develop specialized tissues specific for theproduction of male and female gametes. Successful production of malegametes relies on proper formation of the male reproductive tissues. Thestamen, which embodies the male reproductive organ of plants, containsvarious cell types, including for example, the filament, anther, tapetumand pollen. As used herein, “male tissue” refers to the specializedtissue in a sexually reproducing plant that is responsible forproduction of the male gamete. Male tissues include, but are not limitedto, the stamen, filament, anther, tapetum and pollen.

The process of mature pollen grain formation begins withmicrosporogenesis, wherein meiocytes are formed in the sporogenoustissue of the anther. Microgametogenesis follows, wherein microsporesdivide mitotically and develop into the microgametophyte or pollengrains. The condition of “male fertility” or “male fertile” refers tothose plants producing a mature pollen grain capable of fertilizing afemale gamete to produce a subsequent generation of offspring. The term“influences male fertility” or “modulates male fertility”, as usedherein, refers to any increase or decrease in the ability of a plant toproduce a mature pollen grain when compared to an appropriate control. A“mature pollen grain” or “mature pollen” refers to any pollen graincapable of fertilizing a female gamete to produce a subsequentgeneration of offspring. Likewise, the term “male fertilitypolynucleotide” or “male fertility polypeptide” refers to apolynucleotide or polypeptide that modulates male fertility. A malefertility polynucleotide may, for example, encode a polypeptide thatparticipates in the process of microsporogenesis or microgametogenesis.

Expression of fertility genes has been shown to influence male fertilityin a variety of ways. Mutagenesis studies of Ms22 (also referred to asMsca1) resulted in phenotypically male sterile maize plants with anthersthat did not extrude from the tassel and lacked sporogenous tissue. Westand Albertsen, (1985) Maize Newsletter 59:87; Neuffer, et al., (1977)Mutants of maize. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. See also, U.S. Pat. No. 7,919,676.

Certain male sterility genes such as MAC1, EMS1 or GNE2 (Sorensen, etal., (2002) Plant J. 29:581-594) prevent cell growth in the quartetstage. Mutations in the SPOROCYTELESS/NOZZLE gene act early indevelopment, but impact both anther and ovule formation such that plantsare male and female sterile. The SPOROCYTELESS gene of Arabidopsis isrequired for initiation of sporogenesis and encodes a novel nuclearprotein (Genes Dev. (1999 Aug. 15) 13(16):2108-17).

Ms26 polypeptides have been reported to have significant homology toP450 enzymes found in yeast, plants, and mammals. P450 enzymes have beenwidely studied and characteristic protein domains have been elucidated.The Ms26 protein contains several structural motifs characteristic ofeukaryotic P450's, including the heme-binding domain FxxGxRxCxG (domainD) (SEQ ID NO: 17), domain A A/GGXD/ETT/S (dioxygen-binding) (SEQ ID NO:18), domain B (steroid-binding) and domain C. Phylogenetic tree analysisrevealed that Ms26 is most closely related to P450s involved in fattyacid omega-hydroxylation found in Arabidopsis thaliana and Vicia sativa.See, for example, US Patent Application Publication Number 2012/0005792,herein incorporated by reference.

The Ms45 polynucleotide is a male fertility polynucleotide characterizedin maize (see, for example, U.S. Pat. No. 5,478,369) and wheat (U.S.Provisional Patent Application Ser. No. 61/697,590, filed Sep. 6, 2012).Mutations of Ms45 can result in breakdown of microsporogenesis duringvacuolation of the microspores rendering the mutated plants malesterile. When the cloned Ms45 polynucleotide is introduced into suchmutated male sterile plants, the gene can complement the mutation andconfer male fertility. The cloned Ms45 gene, for example the Ms45 geneof maize or wheat or rice, can also be used to complement male sterilityinduced by expression of pIR molecule targeting the Ms45 promoter, asdescribed herein. Certain embodiments described herein using the MS45gene and/or promoter could be practiced with other genes, such as MS26or Ms22.

Strategies for manipulation of expression of male-fertilitypolynucleotides in wheat will require consideration of the ploidy levelof the individual wheat variety. Triticum aestivum is a hexaploidcontaining three genomes designated A, B, and D (N=21); each genomecomprises seven pairs of nonhomologous chromosomes. Einkorn wheatvarieties are diploids (N=7) and emmer wheat varieties are tetraploids(N=14).

Isolated or substantially purified nucleic acid molecules or proteincompositions are disclosed herein. An “isolated” or “purified” nucleicacid molecule, polynucleotide or protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide or protein asfound in its naturally occurring environment. Thus, an isolated orpurified polynucleotide or protein is substantially free of othercellular material or culture medium when produced by recombinanttechniques or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. A protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, 5% or 1% (by dry weight) ofcontaminating protein. When the polypeptides disclosed herein orbiologically active portion thereof is recombinantly produced, optimallyculture medium represents less than about 30%, 20%, 10%, 5% or 1% (bydry weight) of chemical precursors or non-protein-of-interest chemicals.

A “subject plant” or “subject plant cell” is one in which geneticalteration, such as transformation, has been affected as to a gene ofinterest, or is a plant or plant cell which is descended from a plant orcell so altered and which comprises the alteration. A “control” or“control plant” or “control plant cell” provides a reference point formeasuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or plant cell, i.e., of the same genotype as the starting materialfor the genetic alteration which resulted in the subject plant or cell;(b) a plant or plant cell of the same genotype as the starting materialbut which has been transformed with a null construct (i.e., with aconstruct which has no known effect on the trait of interest, such as aconstruct comprising a marker gene); (c) a plant or plant cell which isa non-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

A. Fragments and Variants of Male Fertility Sequences

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also provided. By “fragment” is intended a portionof the polynucleotide or a portion of the amino acid sequence and henceprotein encoded thereby. Fragments of a polynucleotide may encodeprotein fragments that retain the biological activity of the nativeprotein and hence influence male fertility. Alternatively, fragments ofa polynucleotide that are useful as hybridization probes generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length polynucleotide encoding the polypeptides disclosed herein. Afragment of a promoter polynucleotide may or may not retain promoterfunction. A fragment of a promoter polynucleotide may be used to createa pIR (promoter inverted repeat, aka hairpin) useful in a suppressionconstruct which targets that promoter. See, for example, Matzke, et al.,(2001) Curr. Opin. Genet. Devel. 11:221-227; Mette, et al., (2000) EMBOJ. 19:5194-5201.

A fragment of a polynucleotide that encodes a biologically activeportion of a polypeptide that influences male fertility may encode atleast 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 525or 537 contiguous amino acids, or up to the total number of amino acidspresent in a full-length polypeptide that influences male fertility.Fragments of a polynucleotide encoding a polypeptide that influencesmale fertility that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active portion of a polypeptidethat influences male fertility.

Thus, a fragment of a male fertility polynucleotide as disclosed hereinmay encode a biologically active portion of a male fertility polypeptideor it may be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below or it may be a fragment of apromoter sequence natively associated with a male fertilitypolynucleotide. A biologically active portion of a male fertilitypolypeptide can be prepared by isolating a portion of one of the malefertility polynucleotides disclosed herein, expressing the encodedportion of the male fertility protein (e.g., by recombinant expressionin vitro), and assessing the activity of the encoded portion of the malefertility polypeptide. Polynucleotides that are fragments of a malefertility polynucleotide comprise at least 16, 20, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600 or 1629 nucleotides or up to thenumber of nucleotides present in a full-length male fertilitypolynucleotide.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”or “wild type” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the male fertility polypeptides disclosed herein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant polynucleotides also includesynthetically derived polynucleotides, such as those generated, forexample, by using site-directed mutagenesis but which still encode amale fertility polypeptide. Generally, variants of a particularpolynucleotide disclosed herein will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particular as determinedby sequence alignment programs and parameters described elsewhereherein.

Variants of a particular polynucleotide disclosed herein (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotidesdisclosed herein is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins disclosed herein are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, male fertility activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of a male fertility proteindisclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to the amino acid sequence for the native proteinas determined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a protein disclosedherein may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2or even 1 amino acid residue.

The proteins disclosed herein may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the malefertility polypeptides can be prepared by mutations in the DNA. Methodsfor mutagenesis and polynucleotide alterations are well known in theart. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel, et al., (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff, et al., (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Thus, the genes and polynucleotides disclosed herein include both thenaturally occurring sequences as well as DNA sequence variants whichretain function. Likewise, the male fertility polypeptides and proteinsencompass both naturally occurring polypeptides as well as variationsand modified forms thereof. Such polynucleotide and polypeptide variantswill continue to possess the desired male fertility activity. Themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication Number 75,444.

The deletions, insertions and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assaying for male fertility activity.

Increases or decreases in male fertility can be assayed in a variety ofways. One of ordinary skill in the art can readily assess activity ofthe variant or fragment by introducing the polynucleotide into a planthomozygous for a stable male sterile allele of the polynucleotide, andobserving male tissue development in the plant. In certain embodiments,the variant or fragment polynucleotide is introduced into a plant whichis male-sterile as a result of expression of a polynucleotide whichconfers dominant male sterility. Such a polynucleotide conferringdominant male sterility may be, for example, a pIR directed to thenative promoter of a fertility gene, or a polynucleotide encoding apolypeptide which interferes with development of reproductive tissue,such as DAM-methylase or barnase (See, for example, U.S. Pat. No.5,792,853 or 5,689,049; PCT/EP89/00495).

Variant functional polynucleotides and proteins also encompass sequencesand proteins derived from a mutagenic and recombinogenic procedure suchas DNA shuffling. With such a procedure, one or more different malefertility sequences can be manipulated to create a new male fertilitypolypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the male fertilitypolynucleotides disclosed herein and other known male fertilitypolynucleotides to obtain a new gene coding for a protein with animproved property of interest, such as an increased K, in the case of anenzyme. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) NatureBiotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347;Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri,et al., (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 and5,837,458.

II. Sequence Analysis

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

The use of the term “polynucleotide” is not intended to limit thepresent disclosure to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides disclosed herein also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

III. Expression Cassettes

The male fertility polynucleotides disclosed herein can be provided inexpression cassettes for expression in an organism of interest. Thecassette can include 5′ and 3′ regulatory sequences operably linked to amale fertility polynucleotide as disclosed herein. “Operably linked” isintended to mean a functional linkage between two or more elements. Forexample, an operable linkage between a polynucleotide of interest and aregulatory sequence (e.g., a promoter) is a functional link that allowsfor expression of the polynucleotide of interest. Operably linkedelements may be contiguous or non-contiguous. When used to refer to thejoining of two protein coding regions, by operably linked is intendedthat the coding regions are in the same reading frame.

The expression cassettes disclosed herein may include in the 5′-3′direction of transcription, a transcriptional and translationalinitiation region (i.e., a promoter), a polynucleotide of interest, anda transcriptional and translational termination region (i.e.,termination region) functional in the host cell (i.e., the plant).Expression cassettes are also provided with a plurality of restrictionsites and/or recombination sites for insertion of the male fertilitypolynucleotide to be under the transcriptional regulation of theregulatory regions described elsewhere herein. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the polynucleotide of interest may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the polynucleotide of interest may beheterologous to the host cell or to each other. As used herein,“heterologous” in reference to a polynucleotide or polypeptide sequenceis a sequence that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. As used herein, a chimeric polynucleotide comprises acoding sequence operably linked to a transcription initiation regionthat is heterologous to the coding sequence.

In certain embodiments the polynucleotides disclosed herein can bestacked with any combination of polynucleotide sequences of interest orexpression cassettes as disclosed elsewhere herein. For example, themale fertility polynucleotides disclosed herein may be stacked with anyother polynucleotides encoding male-gamete disruptive polynucleotides orpolypeptides, cytotoxins, markers or other male fertility sequences asdisclosed elsewhere herein. The stacked polynucleotides may be operablylinked to the same promoter as the male fertility polynucleotide, or maybe operably linked to a separate promoter polynucleotide.

As described elsewhere herein, expression cassettes may comprise apromoter operably linked to a polynucleotide of interest, along with acorresponding termination region. The termination region may be nativeto the transcriptional initiation region, may be native to the operablylinked male fertility polynucleotide of interest or with the malefertility promoter sequences, may be native to the plant host, or may bederived from another source (i.e., foreign or heterologous). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144;Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev.5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al.,(1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res.17:7891-7903 and Joshi, et al., (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides of interest may be optimized forincreased expression in the transformed plant. That is, thepolynucleotides can be synthesized using plant-preferred codons forimproved expression. See, for example, Campbell and Gowri, (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing plant-preferred genes. See,for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 and Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats and other such well-characterized sequences thatmay be deleterious to gene expression. The G-C content of the sequencemay be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Johnson, et al., (1986) Virology 154:9-20) and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak, et al.,(1991) Nature 353:90-94); untranslated leader from the coat protein mRNAof alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989)in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) andmaize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991)Virology 81:382-385). See also, Della-Cioppa, et al., (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A. Expression Cassettes Comprising a Male Fertility Polynucleotide

In particular embodiments, the expression cassettes disclosed hereincomprise a promoter operably linked to a male fertility polynucleotide,or active fragment or variant thereof, as disclosed herein. In certainembodiments, a male fertility promoter disclosed herein, or an activefragment or variant thereof, is operably linked to a male fertilitypolynucleotide disclosed herein, or an active fragment or variantthereof.

In certain embodiments, plant promoters can preferentially initiatetranscription in certain tissues, such as stamen, anther, filament andpollen, or developmental growth stages, such as sporogenous tissue,microspores and microgametophyte. Such plant promoters are referred toas “tissue-preferred”, “cell type-preferred” or “growth-stagepreferred”. Promoters which initiate transcription only in certaintissue are referred to as “tissue-specific”. Likewise, promoters whichinitiate transcription only at certain growth stages are referred to as“growth stage-specific”. A “cell type-specific” promoter drivesexpression only in certain cell types in one or more organs, forexample, stamen cells, or individual cell types within the stamen suchas anther, filament or pollen cells.

Male fertility polynucleotides disclosed herein, and active fragmentsand variants thereof, can be operably linked to male-tissue-specific ormale-tissue-preferred promoters including, for example, stamen-specificor stamen-preferred promoters, anther-specific or anther-preferredpromoters, pollen-specific or pollen-preferred promoters,tapetum-specific promoters or tapetum-preferred promoters, and the like.Promoters can be selected based on the desired outcome. For example, thepolynucleotides of interest can be operably linked to constitutive,tissue-preferred, growth stage-preferred or other promoters forexpression in plants.

In one embodiment, the promoters may be those which preferentiallyexpress a polynucleotide of interest in the male tissues of the plant.No particular male fertility tissue-preferred promoter must be used inthe process, and any of the many such promoters known to one skilled inthe art may be employed. One such promoter is the 5126 promoter, whichpreferentially directs expression of the polynucleotide to which it islinked to male tissue of the plants, as described in U.S. Pat. Nos.5,837,851 and 5,689,051. Other examples include the maize Ms45 promoterdescribed at U.S. Pat. No. 6,037,523; SF3 promoter described at U.S.Pat. No. 6,452,069; the BS92-7 promoter described at WO 2002/063021; aSGB6 regulatory element described at U.S. Pat. No. 5,470,359; the TA29promoter (Koltunow, et al., (1990) Plant Cell 2:1201-1224; Goldberg, etal., (1993) Plant Cell 5:1217-1229 and U.S. Pat. No. 6,399,856); thetype 2 metallothionein-like gene promoter (Charbonnel-Campaa, et al.,(2000) Gene 254:199-208) and the Brassica Bca9 promoter (Lee, et al,(2003) Plant Cell Rep. 22:268-273).

In some embodiments, expression cassettes comprise male-gamete-preferredpromoters operably linked to a male fertility polynucleotide.Male-gamete-preferred promoters include the PG47 promoter (U.S. Pat. No.5,412,085; U.S. Pat. No. 5,545,546; Plant J 3(2):261-271 (1993)), aswell as ZM13 promoter (Hamilton, et al., (1998) Plant Mol. Biol.38:663-669); actin depolymerizing factor promoters (such as Zmabp1,Zmabp2; see, for example, Lopez, et al, (1996) Proc. Natl. Acad. Sci.USA 93:7415-7420); the promoter of the maize pectin methylesterase-likegene, ZmC5 (Wakeley, et al., (1998) Plant Mol. Biol. 37:187-192); theprofilin gene promoter Zmpro1 (Kovar, et al., (2000) The Plant Cell12:583-598); the sulphated pentapeptide phytosulphokine gene ZmPSK1(Lorbiecke, et al., (2005) Journal of Experimental Botany56(417):1805-1819); the promoter of the calmodulin binding protein Mpcbp(Reddy, et al., (2000) J. Biol. Chem. 275(45):35457-70).

As disclosed herein, constitutive promoters include, for example, thecore promoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; the core CaMV35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin(McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen,et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al.,(1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor.Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J.3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like.Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142 and 6,177,611.

“Seed-preferred” promoters include both those promoters active duringseed development such as promoters of seed storage proteins as well asthose promoters active during seed germination. See, Thompson, et al.,(1989) BioEssays 10:108, herein incorporated by reference. Suchseed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase) (see, WO 2000/11177 and U.S. Pat.No. 6,225,529, herein incorporated by reference). Gamma-zein is anendosperm-specific promoter. Globulin-1 (Glob-1) is a representativeembryo-specific promoter. For dicots, seed-specific promoters include,but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybeanlectin, cruciferin, and the like. For monocots, seed-specific promotersinclude, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDazein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. Seealso, WO 2000/12733, where seed-preferred promoters from end1 and end2genes are disclosed; herein incorporated by reference. Additional embryospecific promoters are disclosed in Sato, et al., (1996) Proc. Natl.Acad. Sci. 93:8117-8122; Nakase, et al., (1997) Plant J 12:235-46 andPostma-Haarsma, et al., (1999) Plant Mol. Biol. 39:257-71. Additionalendosperm specific promoters are disclosed in Albani, et al., (1984)EMBO 3:1405-15; Albani, et al., (1999) Theor. Appl. Gen. 98:1253-62;Albani, et al., (1993) Plant J. 4:343-55; Mena, et al., (1998) The PlantJournal 116:53-62 and Wu, et al., (1998) Plant Cell Physiology39:885-889.

Dividing cell or meristematic tissue-preferred promoters have beendisclosed in Ito, et al., (1994) Plant Mol. Biol. 24:863-878; Reyad, etal., (1995) Mo. Gen. Genet. 248:703-711; Shaul, et al., (1996) Proc.Natl. Acad. Sci. 93:4868-4872; Ito, et al., (1997) Plant J. 11:983-992;and Trehin, et al., (1997) Plant Mol. Biol. 35:667-672.

Stress inducible promoters include salt/water stress-inducible promoterssuch as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89);cold-inducible promoters, such as, cor15a (Hajela, et al., (1990) PlantPhysiol. 93:1246-1252), cor15b (Wlihelm, et al., (1993) Plant Mol Biol23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 423-324-328),ci7 (Kirch, et al., (1997) Plant Mol Biol. 33:897-909), ci21A(Schneider, et al., (1997) Plant Physiol. 113:335-45); drought-induciblepromoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol.30:1247-57), rd29 (Kasuga, et al., (1999) Nature Biotechnology18:287-291); osmotic inducible promoters, such as, Rab17 (Vilardell, etal., (1991) Plant Mol. Biol. 17:985-93) and osmotin (Raghothama, et al.,(1993) Plant Mol Biol 23:1117-28) and heat inducible promoters, such as,heat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75; Marrs,et al., (1993) Dev. Genet. 14:27-41) and smHSP (Waters, et al., (1996)J. Experimental Botany 47:325-338). Other stress-inducible promotersinclude rip2 (U.S. Pat. No. 5,332,808 and US Patent ApplicationPublication Number 2003/0217393) and rp29a (Yamaguchi-Shinozaki, et al.,(1993) Mol. Gen. Genetics 236:331-340).

As discussed elsewhere herein, the expression cassettes comprising malefertility polynucleotides may be stacked with other polynucleotides ofinterest. Any polynucleotide of interest may be stacked with the malefertility polynucleotide, including for example, male-gamete-disruptivepolynucleotides and marker polynucleotides.

Male fertility polynucleotides disclosed herein may be stacked in orwith expression cassettes comprising a promoter operably linked to apolynucleotide which is male-gamete-disruptive; that is, apolynucleotide which interferes with the function, formation, ordispersal of male gametes. A male-gamete-disruptive polynucleotide canoperate to prevent function, formation, or dispersal of male gametes byany of a variety of methods. By way of example but not limitation, thiscan include use of polynucleotides which encode a gene product such asDAM-methylase or barnase (see, for example, U.S. Pat. No. 5,792,853 or5,689,049; PCT/EP89/00495); encode a gene product which interferes withthe accumulation of starch or affects osmotic balance in pollen (see,for example, U.S. Pat. Nos. 7,875,764; 8,013,218; 7,696,405); inhibitformation of a gene product important to male gamete function, formationor dispersal (see, for example, U.S. Pat. Nos. 5,859,341; 6,297,426);encode a gene product which combines with another gene product toprevent male gamete formation or function (see, U.S. Pat. Nos.6,162,964; 6,013,859; 6,281,348; 6,399,856; 6,248,935; 6,750,868;5,792,853); are antisense to, or cause co-suppression of, a genecritical to male gamete function, formation, or dispersal (see, U.S.Pat. Nos. 6,184,439; 5,728,926; 6,191,343; 5,728,558; 5,741,684);interfere with expression of a male fertility polynucleotide through useof hairpin formations (Smith, et al., (2000) Nature 407:319-320; WO1999/53050 and WO 1998/53083; Matzke, et al., (2001) Curr. Opin. Genet.Devel. 11:221-227;); see also, Scheid, et al., (2002) Proc. Natl. Acad.Sci., USA 99:13659-13662; Waterhouse and Helliwell, (2003) NatureReviews Genetics 4:29-38; Aufsaftz, et al., (2002) Proc. Nat'l. Acad.Sci. 99(4):16499-16506; Sijen, et al., (2001) Curr. Biol. 11:436-440 orthe like.

Male-gamete-disruptive polynucleotides include dominant negative genessuch as methylase genes and growth-inhibiting genes. See, U.S. Pat. No.6,399,856. Dominant negative genes include diphtheria toxin A-chain gene(Czako and An, (1991) Plant Physiol. 95:687-692; Greenfield, et al.,(1983) PNAS 80:6853); cell cycle division mutants such as CDC in maize(Colasanti, et al., (1991) PNAS 88:3377-3381); the WT gene (Farmer, etal., (1994) Mol. Genet. 3:723-728) and P68 (Chen, et al., (1991) PNAS88:315-319).

Further examples of male-gamete-disruptive polynucleotides include, butare not limited to, pectate lyase gene pelE from Erwinia chrysanthermi(Kenn, et al., (1986) J. Bacteriol. 168:595); CytA toxin gene fromBacillus thuringiensis Israeliensis (McLean, et al., (1987) J.Bacteriol. 169:1017, U.S. Pat. No. 4,918,006); DNAses, RNAses, proteasesor polynucleotides expressing anti-sense RNA. A male-gamete-disruptivepolynucleotide may encode a protein involved in inhibiting pollen-stigmainteractions, pollen tube growth, fertilization, or a combinationthereof.

Male fertility polynucleotides disclosed herein may be stacked withexpression cassettes disclosed herein comprising a promoter operablylinked to a polynucleotide of interest encoding a reporter or markerproduct. Examples of suitable reporter polynucleotides known in the artcan be found in, for example, Jefferson, et al., (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell. Biol. 7:725-737;Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al., (1995)BioTechniques 19:650-655 and Chiu, et al., (1996) Current Biology6:325-330. In certain embodiments, the polynucleotide of interestencodes a selectable reporter. These can include polynucleotides thatconfer antibiotic resistance or resistance to herbicides. Examples ofsuitable selectable marker polynucleotides include, but are not limitedto, genes encoding resistance to chloramphenicol, methotrexate,hygromycin, streptomycin, spectinomycin, bleomycin, sulfonamide,bromoxynil, glyphosate and phosphinothricin.

In some embodiments, the expression cassettes disclosed herein comprisea polynucleotide of interest encoding scorable or screenable markers,where presence of the polynucleotide produces a measurable product.Examples include a β-glucuronidase, or uidA gene (GUS), which encodes anenzyme for which various chromogenic substrates are known (for example,U.S. Pat. Nos. 5,268,463 and 5,599,670); chloramphenicol acetyltransferase, and alkaline phosphatase. Other screenable markers includethe anthocyanin/flavonoid polynucleotides including, for example, aR-locus polynucleotide, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues, thegenes which control biosynthesis of flavonoid pigments, such as themaize C1 and C2, the B gene, the p1 gene, and the bronze locus genes,among others. Further examples of suitable markers encoded bypolynucleotides of interest include the cyan fluorescent protein (CYP)gene, the yellow fluorescent protein gene, a lux gene, which encodes aluciferase, the presence of which may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry, a green fluorescent protein (GFP) and DsRed2(Clontechniques, 2001) where plant cells transformed with the markergene are red in color, and thus visually selectable. Additional examplesinclude a p-lactamase gene encoding an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin), a xylE gene encoding a catechol dioxygenase that canconvert chromogenic catechols, an α-amylase gene and a tyrosinase geneencoding an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone, which in turn condenses to form the easily detectablecompound melanin.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su, et al., (2004)Biotechnol Bioeng 85:610-9 and Fetter, et al., (2004) Plant Cell16.215-28), cyan florescent protein (CYP) (Bolte, et al., (2004) J. CellScience 117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42)and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte, et al.,(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511;Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol.6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, etal., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612;Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc.Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl.Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg;Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow,et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992)Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc.Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) NucleicAcids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc.Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. AgentsChemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg;Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva,et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al.,(1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag,Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures areherein incorporated by reference. The above list of selectable markergenes is not meant to be limiting. Any selectable marker gene can beused in the compositions and methods disclosed herein.

In some embodiments, the expression cassettes disclosed herein comprisea first polynucleotide of interest encoding a male fertilitypolynucleotide operably linked to a first promoter polynucleotide,stacked with a second polynucleotide of interest encoding amale-gamete-disruptive gene product operably linked to a maletissue-preferred promoter polynucleotide. In other embodiments, theexpression cassettes described herein may also be stacked with a thirdpolynucleotide of interest encoding a marker polynucleotide operablylinked to a third promoter polynucleotide.

In specific embodiments, the expression cassettes disclosed hereincomprise a first polynucleotide of interest encoding a wheat malefertility gene disclosed herein operably linked to a promoter, which maybe a tissue-preferred or constitutive promoter, such as the cauliflowermosaic virus (CaMV) 35S promoter. The expression cassettes may furthercomprise a second polynucleotide of interest encoding amale-gamete-disruptive gene product operably linked to a maletissue-preferred promoter. In certain embodiments, the expressioncassettes disclosed herein may further comprise a third polynucleotideof interest encoding a marker gene, such as the phosphinothricinacetyltransferase (PAT) gene from Streptomyces viridochomagenes operablylinked to a constitutive promoter, such as the cauliflower mosaic virus(CaMV) 35S promoter.

IV. Plants

A. Plants Having Altered Levels/Activity of Male Fertility Polypeptide

Further provided are plants having altered levels and/or activities of amale fertility polypeptide and/or altered levels of male fertility. Insome embodiments, the plants disclosed herein have stably incorporatedinto their genomes a heterologous male fertility polynucleotide, oractive fragments or variants thereof, as disclosed herein. Thus, plants,plant cells, plant parts, and seeds are provided which comprise at leastone heterologous male fertility polynucleotide as set forth in any oneof SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any usefulfragments or variants disclosed herein.

Plants are further provided comprising the expression cassettesdisclosed herein comprising a male fertility polynucleotide operablylinked to a promoter that is active in the plant. In some embodiments,expression of the male fertility polynucleotide modulates male fertilityof the plant. In certain embodiments, expression of the male fertilitypolynucleotide increases male fertility of the plant. For example,plants are provided comprising an expression cassette comprising an MS45polynucleotide as set forth in SEQ ID NO: 8, or an active fragment orvariant thereof, operably linked to a promoter. Upon expression of theMs45 polynucleotide, male fertility of the plant is increased.

In certain embodiments, expression cassettes comprising a heterologousmale fertility polynucleotide as disclosed herein, or an active fragmentor variant thereof, operably linked to a promoter active in a plant, areprovided to a male sterile plant. Upon expression of the heterologousmale fertility polynucleotide, the male fertility of the plant isrestored. In specific embodiments, the plants disclosed herein comprisean expression cassette comprising a heterologous male fertilitypolynucleotide as disclosed herein, or an active fragment or variantthereof, operably linked to a promoter, stacked with one or moreexpression cassettes comprising a polynucleotide of interest operablylinked to a promoter active in the plant. For example, the stackedpolynucleotide of interest can comprise a male-gamete-disruptivepolynucleotide and/or a marker polynucleotide.

Plants disclosed herein may also comprise stacked expression cassettesdescribed herein comprising at least two polynucleotides such that theat least two polynucleotides are inherited together in more than 50% ofmeioses, i.e., not randomly. Accordingly, when a plant or plant cellcomprising stacked expression cassettes with two polynucleotidesundergoes meiosis, the two polynucleotides segregate into the sameprogeny (daughter) cell. In this manner, stacked polynucleotides willlikely be expressed together in any cell for which they are present. Forexample, a plant may comprise an expression cassette comprising a malefertility polynucleotide stacked with an expression cassette comprisinga male-gamete-disruptive polynucleotide such that the male fertilitypolynucleotide and the male-gamete-disruptive polynucleotide areinherited together. Specifically, a male sterile plant could comprise anexpression cassette comprising a male fertility polynucleotide disclosedherein operably linked to a constitutive promoter, stacked with anexpression cassette comprising a male-gamete-disruptive polynucleotideoperably linked to a male tissue-preferred promoter, such that the plantproduces mature pollen grains. However, in such a plant, development ofthe daughter pollen cells comprising the male fertility polynucleotidewill be impacted by expression of the male-gamete-disruptivepolynucleotide.

B. Plants and Methods of Introduction

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which a plant can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, grain and the like. As used herein “grain” is intended themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants and mutants of theregenerated plants are also included within the scope of the disclosure,provided that these parts comprise the introduced nucleic acidsequences.

The methods disclosed herein comprise introducing a polypeptide orpolynucleotide into a plant cell. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell. Themethods disclosed herein do not depend on a particular method forintroducing a sequence into the host cell, only that the polynucleotideor polypeptides gains access to the interior of at least one cell of thehost. Methods for introducing polynucleotide or polypeptides into hostcells (i.e., plants) are known in the art and include, but are notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods. In some embodiments, apolynucleotide is introduced to a plant by sexual cross to anotherplant. For example, pollen comprising a polynucleotide of interest istransferred to the stigma of a receptor plant, to produce progenycomprising the polynucleotide of interest.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a host (i.e., a plant) integrates into thegenome of the plant and is capable of being inherited by the progenythereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the host (i.e., a plant) and expressedtemporally or a polypeptide is introduced into a host (i.e., a plant).

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway, etal., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al.,(1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et al.,U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski, et al.,(1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see,for example, Sanford, et al., U.S. Pat. No. 4,945,050; Tomes, et al.,U.S. Pat. No. 5,879,918; Tomes, et al., U.S. Pat. No. 5,886,244; Bidney,et al., U.S. Pat. No. 5,932,782; Tomes, et al., (1995) “Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment,” inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); McCabe, et al., (1988)Biotechnology 6:923-926) and Lec1 transformation (WO 2000/28058). Alsosee, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, etal., (1987) Particulate Science and Technology 5:27-37 (onion);Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, etal., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen,(1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al.,(1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990)Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad.Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising, et al., U.S.Pat. Nos. 5,322,783 and 5,324,646; Tomes, et al., (1995) “Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment,” inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg(Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol.91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier,et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); DeWet, et al., (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen);Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, etal., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediatedtransformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505(electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 andChristou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, etal., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacteriumtumefaciens), all of which are herein incorporated by reference.

Wheat transformation protocols are available to one of skill in the art.See, for example, He, et al., (2010) J. Exp. Botany 61(6):1567-1581; Wu,et al., (2008) Transgenic Res. 17:425-436; Nehra, et al., (1994) PlantJ. 5(2):285-297; Rasco-Gaunt, et al., (2001) J. Exp. Botany52(357):865-874; Razzaq, et al., (2011) African J. Biotech.10(5):740-750. See also, Tamás-Nyitrai, et al., (2012) Plant CellCulture Protocols, Methods in Molecular Biology 877:357-384.

In specific embodiments, the male fertility polynucleotides orexpression cassettes disclosed herein can be provided to a plant using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, the introductionof the male fertility polypeptide or variants and fragments thereofdirectly into the plant or the introduction of a male fertilitytranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway, etal., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) PlantSci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the male fertility polynucleotide or expression cassettesdisclosed herein can be transiently transformed into the plant usingtechniques known in the art. Such techniques include viral vector systemand the precipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, the transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

In other embodiments, the male fertility polynucleotides or expressioncassettes disclosed herein may be introduced into plants by contactingplants with a virus or viral nucleic acids. Generally, such methodsinvolve incorporating a nucleotide construct of disclosed herein withina viral DNA or RNA molecule. It is recognized that a male fertilitysequence disclosed herein may be initially synthesized as part of aviral polyprotein, which later may be processed by proteolysis in vivoor in vitro to produce the desired recombinant protein. Methods forintroducing polynucleotides into plants and expressing a protein encodedtherein, involving viral DNA or RNA molecules, are known in the art.See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931 and Porta, et al., (1996) Molecular Biotechnology5:209-221, herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide disclosed herein can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. These are referred to as T0 plants.See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84.These plants may then be grown, and pollinated with either the sametransformed strain or different strains, and the resulting progenyhaving desired expression of the desired phenotypic characteristicidentified. Two or more generations (e.g., T1, T2, T3) may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present disclosure provides transformed seed (alsoreferred to as “transgenic seed”) having a male fertility polynucleotidedisclosed herein, for example, an expression cassette disclosed herein,stably incorporated into their genome. Seed comprising any expressioncassette disclosed herein can be sorted based on size parameters,including but not limited to, seed length, seed width, seed density,seed color, or any combination thereof.

The male fertility polynucleotides and expression cassettes disclosedherein may be used for transformation of any plant species, including,but not limited to, monocots and dicots. Examples of plant species ofinterest include, but are not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, grasses and conifers.

In particular embodiments, wheat plants are used in the methods andcompositions disclosed herein. As used herein, the term “wheat” refersto any species of the genus Triticum, including progenitors thereof, aswell as progeny thereof produced by crosses with other species. Wheatincludes “hexaploid wheat” which has genome organization of AABBDD,comprised of 42 chromosomes, and “tetraploid wheat” which has genomeorganization of AABB, comprised of 28 chromosomes. Hexaploid wheatincludes T. aestivum, T spelta, T. mocha, T. compactum, T.sphaerococcum, T. vavilovii and interspecies cross thereof. Tetraploidwheat includes T. durum (also referred to as durum wheat or Triticumturgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum andinterspecies cross thereof. In addition, the term “wheat” includespossible progenitors of hexaploid or tetraploid Triticum sp. such as T.uartu, T. monococcum or T. boeoticum for the A genome, Aegilopsspeltoides for the B genome, and T. tauschii (also known as Aegilopssquarrosa or Aegilops tauschii) for the D genome. A wheat cultivar foruse in the present disclosure may belong to, but is not limited to, anyof the above-listed species. Also encompassed are plants that areproduced by conventional techniques using Triticum sp. as a parent in asexual cross with a non-Triticum species, such as rye Secale cereale,including but not limited to Triticale. In some embodiments, the wheatplant is suitable for commercial production of grain, such as commercialvarieties of hexaploid wheat or durum wheat, having suitable agronomiccharacteristics which are known to those skilled in the art.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima) and chrysanthemum.

Conifers that may be employed in practicing the present methods andcompositions include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta) and Monterey pine (Pinus radiata);Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea) andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants disclosedherein are crop plants (for example, corn, alfalfa, sunflower, Brassica,soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco,etc.). In other embodiments, corn and soybean plants are optimal and inyet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Typically, an intermediate host cell will be used in the practice of themethods and compositions disclosed herein to increase the copy number ofthe cloning vector. With an increased copy number, the vector containingthe nucleic acid of interest can be isolated in significant quantitiesfor introduction into the desired plant cells. In one embodiment, plantpromoters that do not cause expression of the polypeptide in bacteriaare employed.

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.8:4057) and the lambda derived P L promoter and N-gene ribosome bindingsite (Shimatake, et al., (1981) Nature 292:128). The inclusion ofselection markers in DNA vectors transfected in E coli. is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein disclosed herein areavailable using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene22:229-235); Mosbach, et al., (1983) Nature 302:543-545).

In some embodiments, the expression cassette or male fertilitypolynucleotides disclosed herein are maintained in a hemizygous state ina plant. Hemizygosity is a genetic condition existing when there is onlyone copy of a gene (or set of genes) with no allelic counterpart on thesister chromosome. In certain embodiments, the expression cassettesdisclosed herein comprise a first promoter operably linked to a malefertility polynucleotide which is stacked with a male-gamete-disruptivepolynucleotide operably linked to a male tissue-preferred promoter, andsuch expression cassettes are introduced into a male sterile plant in ahemizygous condition. When the male fertility polynucleotide isexpressed, the plant is able to successfully produce mature pollengrains because the male fertility polynucleotide restores the plant to afertile condition. Given the hemizygous condition of the expressioncassette, only certain daughter cells will inherit the expressioncassette in the process of pollen grain formation. The daughter cellsthat inherit the expression cassette containing the male fertilitypolynucleotide will not develop into mature pollen grains due to themale tissue-preferred expression of the stacked encodedmale-gamete-disruptive gene product. Those pollen grains that do notinherit the expression cassette will continue to develop into maturepollen grains and be functional, but will not contain the male fertilitypolynucleotide of the expression cassette and therefore will nottransmit the male fertility polynucleotide to progeny through pollen.

V. Modulating the Concentration and/or Activity of Male FertilityPolypeptides

A method for modulating the concentration and/or activity of the malefertility polypeptides disclosed herein in a plant is provided. The term“influences” or “modulates”, as used herein with reference to theconcentration and/or activity of the male fertility polypeptides, refersto any increase or decrease in the concentration and/or activity of themale fertility polypeptides when compared to an appropriate control. Ingeneral, concentration and/or activity of a male fertility polypeptidedisclosed herein is increased or decreased by at least 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a native control plant,plant part, or cell. Modulation as disclosed herein may occur duringand/or subsequent to growth of the plant to the desired stage ofdevelopment. In specific embodiments, the male fertility polypeptidesdisclosed herein are modulated in monocots, particularly wheat.

A variety of methods can be employed to assay for modulation in theconcentration and/or activity of a male fertility polypeptide. Forinstance, the expression level of the male fertility polypeptide may bemeasured directly, for example, by assaying for the level of the malefertility polypeptide or RNA in the plant (i.e., Western or Northernblot), or indirectly, for example, by assaying the male fertilityactivity of the male fertility polypeptide in the plant. Methods formeasuring the male fertility activity are described elsewhere herein. Inspecific embodiments, modulation of male fertility polypeptideconcentration and/or activity comprises the modulation (i.e., anincrease or a decrease) in the level of male fertility polypeptide inthe plant. Methods to measure the level and/or activity of malefertility polypeptides are known in the art and are discussed elsewhereherein. In still other embodiments, the level and/or activity of themale fertility polypeptide is modulated in vegetative tissue, inreproductive tissue, or in both vegetative and reproductive tissue.

In one embodiment, the activity and/or concentration of the malefertility polypeptide is increased by introducing the polypeptide or thecorresponding male fertility polynucleotide into the plant.Subsequently, a plant having the introduced male fertility sequence isselected using methods known to those of skill in the art such as, butnot limited to, Southern blot analysis, DNA sequencing, PCR analysis orphenotypic analysis. In certain embodiments, marker polynucleotides areintroduced with the male fertility polynucleotide to aid in selection ofa plant having or lacking the male fertility polynucleotide disclosedherein. A plant or plant part altered or modified by the foregoingembodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of the malefertility polypeptide in the plant. Plant forming conditions are wellknown in the art.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, or introducing into theplant (transiently or stably) a polynucleotide construct encoding a malefertility polypeptide. It is also recognized that the methods disclosedherein may employ a polynucleotide that is not capable of directing, inthe transformed plant, the expression of a protein or an RNA. Thus, thelevel and/or activity of a male fertility polypeptide may be increasedby altering the gene encoding the male fertility polypeptide or itspromoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868. Therefore mutagenized plants that carry mutations inmale fertility genes, where the mutations increase expression of themale fertility gene or increase the activity of the encoded malefertility polypeptide are provided.

In other embodiments, the concentration and/or activity of a malefertility polypeptide is increased by introduction into a plant of anexpression cassette comprising a male fertility polynucleotide (e.g. SEQID NO: 8 or 10), or an active fragment or variant thereof, as disclosedelsewhere herein. The male fertility polynucleotide may be operablylinked to promoter that is heterologous to the plant or native to theplant. By increasing the concentration and/or activity of a malefertility polypeptide in a plant, the male fertility of the plant islikewise increased. Thus, the male fertility of a plant can be increasedby increasing the concentration and/or activity of a male fertilitypolypeptide. For example, male fertility can be restored to a malesterile plant by increasing the concentration and/or activity of a malefertility polypeptide.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or anRNA. For example, the polynucleotides disclosed herein may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, allof which are herein incorporated by reference. See also, WO 1998/49350,WO 1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl.Acad. Sci. USA 96:8774-8778, herein incorporated by reference. It istherefore recognized that methods disclosed herein do not depend on theincorporation of the entire polynucleotide into the genome, only thatthe plant or cell thereof is altered as a result of the introduction ofthe polynucleotide into a cell.

In one embodiment, the genome may be altered following the introductionof the polynucleotide into a cell. For example, the polynucleotide, orany part thereof, may incorporate into the genome of the plant.Alterations to the genome disclosed herein include, but are not limitedto, additions, deletions, and substitutions of nucleotides into thegenome. While the methods disclosed herein do not depend on additions,deletions and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions or substitutions comprisesat least one nucleotide.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisdisclosure pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

TABLE 1 Summary of SEQ ID NOS SEQ ID: Description 1 Wheat MS45promoter_4AL 2 Wheat MS45 promoter_4BS 3 Wheat MS45 promoter_4DL 4 WheatMS45 promoter consensus 5 Maize MS45 promoter fragment 6 Wheat pIR 7 PHP54693 T-DNA 8 Rice MS45 genomic region used in PHP37034 9 Maize MS45promoter region used in PHP 37034 10 Rice MS26 cds 11 Plant-optimizedDAM 12 PHP 56791 T-DNA 13 PHP 54783 T-DNA 14 Maize/wheat consensus ofFIG. 2 15 Wheat PRO/pIR consensus of FIG. 3 16 Full-length consensus ofFIGS. 1A and 1B

EXPERIMENTAL Example 1. Identification of Wheat MS45 Regulatory Region

This example demonstrates the identification of the wheat DNA sequencesthat correspond to elements to control expression of wheat MS45 inplanta.

The 413 amino acid sequence of Zea mays MS45 (Cigan, et al., (2001) SexPlant Reprod. 14:135-142) was used to search the wheat genomic 454sequences, CerealsDB, (Wilkenson, et al., (2012) BMC Bioinformatics13:219) of Chinese Spring wheat to identify and assembly contigscontaining wheat MS45 ortholog sequences. Three non-identical contigswere assembled of approximately 1000 nucleotides, corresponding tosequences from 4AL, 4BS and 4DL corresponding hexaploid wheat Triticumaestivum (SEQ ID NOS: 1, 2 and 3). Alignment of theses sequences revealshigh sequence identity across the three contigs from wheat (FIG. 1).Similarly, alignment of a 500 bp region containing a consensus sequenceof the wheat promoters (SEQ ID NO: 4) with the 500 bp region (SEQ ID NO:5) containing the ZmMS45 promoter region shows regions of similarityextending to nearly 73% identity across nucleotide positions 369-426 ofthe wheat and maize sequences, suggesting conservation of regulatoryelements between wheat and maize. Overall, 45% sequence identity isobserved across the entire 500 base pair region of wheat and maize (FIG.2).

A synthetic DNA sequence (SEQ ID NO: 6) was generated that contains 98%sequence identity to the wheat Ms45 consensus (FIG. 3). The syntheticwheat Ms45 promoter inverted repeat sequence of SEQ ID NO: 6 was used ingene suppression studies described in examples below.

Example 2. Promoter-Inverted-Repeat Expression Affects Plant Fertilityin Wheat

This example demonstrates that the fertility or fertility potential ofplants can be altered by expression of promoter inverted repeatmolecules (pIR) specific for the promoter of a gene that encodes aprotein involved in male fertility pathway.

A promoter inverted repeat construct was generated by linking aubiquitin promoter to inverted repeats which contained a portion of thewheat MS45 promoter (SEQ ID NO: 6), including a NOS spacer segmentbetween the inverted repeat sequences. Nucleic acid molecules andmethods for preparing the vector PHP54693 were as previously described(Cigan, et al., (2005) Plant Journal 43:929-940). SEQ ID NO: 7 containsthe T-DNA sequence for PHP54693. PHP54693 was introduced into wheatFielder variety by Agrobacterium-mediated transformation using methodsknown in the art and referenced elsewhere herein.

Plants were grown in the greenhouse; transgene copy-number wasdetermined by quantitative polymerase chain reaction (QPCR). Plants weregrown to maturity and male fertility phenotype was recorded. Results areshown in Table 2.

TABLE 2 Male Fertility phenotype of transgenic wheat plants containingPHP54693. TOTAL SINGLE OR MULTI- PHP54693 EVENTS LOW COPY COPY MALESTERILE 36 20 16 MALE FERTILE 13 8 5 49

Suppression was sufficient to cause male-sterility in 73% of events.Both single-copy and multi-copy T-DNA insertion events weremale-sterile, at approximately equal frequency, indicating that bothsingle-copy and multi-copy insertion events are effective.

Microscopic examination of anthers from several independent PHP54693plants revealed that these anthers lacked pollen in contrast tosimilarly staged anthers from untransformed Fielder plants. In addition,microspores isolated from anthers of male sterile PHP54693 plants wereobserved to break down after the quartet stage of development. Thisobservation is similar to the stage at which microspores from malesterile maize ms45 mutants are observed to break down. These resultsdemonstrate that a pIR construct directed to wheat MS45 promoter iscapable of generating male sterile wheat plants.

It is noted that the pIR of PHP54693 is driven by a constitutive,heterologous promoter, i.e. ZmUBI. This demonstrates that one of skillin the art may select from among a wide range of promoters for use inthe suppression construct, including any promoter which providesexpression in the tissue wherein the target gene is expressed and inwhich suppression is desired. In certain embodiments the promoter maydrive expression preferentially in one or more male reproductivetissues.

Example 3. Expression of Exogenous MS45 Gene Product Restores Fertility

This example demonstrates that male-sterile plants containing a pIRconstruct targeting the wheat MS45 promoter (PHP54693 T-DNA, SEQ ID NO:7) can be restored to male fertility when also containing an exogenousMS45 gene construct.

Constructs were prepared containing an MS45 coding sequence derived fromrice (SEQ ID NO: 8) operably linked to a heterologous maize Ms45promoter (SEQ ID NO: 9). This construct was introduced into wheatFielder variety by Agrobacterium-mediated transformation as describedabove. Regenerated transformed wheat plants were grown in thegreenhouse. All regenerated PHP37034 wheat plants were male fertile.

A wheat plant containing a single-copy PHP37034 TDNA insertion (Male 1)was used as a pollen donor and crossed onto two non-identical malesterile PHP54693 plants (Female 1 and Female 2). Seed was harvested fromthese crosses, planted, and progeny genotyped for the presence ofPHP54693 and PHP37034 TDNA insertions by PCR. Plants containing onlyPHP54693 or both TDNAs, PHP54693 and PHP37034, were grown to maturityand male fertility phenotype recorded.

As shown in Table 3, Group 1 and 2 wheat plants containing only PHP54693did not contain pollen and were male sterile (No Seed). In contrast,PHP54693 plants also containing PHP37034 from both groups shed pollenand were capable of self-fertilization (Seed). Seed number per plant inPHP54693/PHP37034 progeny was similar to seed numbers obtained fromuntransformed Fielder variety plants.

TABLE 3 Male fertility phenotype of transgenic wheat plants containingDominant sterility construct PHP54693 and Restorer PHP37034. DominantSterility Construct RESTORER PLANT GROUP PHP54693 PHP37034 FEMALE MALESEED SET 1 1 + + 1 1 SEED 2 1 + + 1 1 SEED 3 1 + + 1 1 SEED 4 1 + 1 1 NOSEED 5 1 + 1 1 NO SEED 6 1 + 1 1 NO SEED 1 2 + + 2 1 SEED 2 2 + + 2 1SEED 3 2 + + 2 1 SEED 4 2 + + 2 1 NO SEED 5 2 + 2 1 NO SEED 6 2 + 2 1 NOSEED

These data provide the surprising result that in hexaploid Fielderwheat, the A, B and D genome copies of the wheat Ms45 promoter aresuppressed by PHP54693, resulting in loss of Ms45 expression and malesterile wheat. These results further demonstrate that an exogenous MS45gene construct contained in PHP37034 is capable of restoring fertilityto hexaploid wheat plants containing the Dominant male sterilityconstruct PHP54693 which suppresses the endogenous wheat MS45 gene.

Example 4. Use of Exogenous MS45 Gene Products to Restore Fertility inPHP54693 Plants

The promoter expressing the rice MS45 gene in PHP37034 can be derivedfrom a source other than maize; for example, the rice and Arabidopsishomologs of the maize MS45, 5126, BS7 and MS26 genes, can be used, orany plant promoter capable of transcribing MS45 such that expression ofthe transcription unit renders plants male fertile, including aconstitutive promoter. In certain respects, it is advantageous to usenon-wheat promoters to express the fertility-restoring gene, such as theMS45 gene. For example, where promoter inverted repeats from the samespecies reduce target gene function such that the plant is non-viable ornon-reproductive, a promoter from a different species can be used totranscriptionally express the complementing gene function (e.g., MS45),thus circumventing this potential problem. Alternatively, promotersnatively associated with genes other than MS45 may be used, providedthat the expression occurs at least in tissues in which complementationis desired, including male-tissue-preferred or constitutive promotersfrom wheat or from other species. Further, a native promoter, forexample a wheat MS45 promoter, can be used to drive thefertility-restoring gene if that native promoter is sufficiently alteredthat it is not targeted by the pIR.

In addition, the MS45 gene in PHP37034 can be from a source other thanrice, for example the maize or wheat MS45 coding region.

Taken together, the present Examples demonstrate that an endogenouspolyploid plant fertility gene can be inactivated using promoterinverted repeat-mediated suppression, and that a fertile phenotype canbe restored in genotypically sterile plants.

Example 5. Inbred Maintenance and Increase of LOF-plRmf Male SterilePlants Using a Hemizygous Maintainer

It would be advantageous to produce a pure line of male sterile plantsto allow for cross pollination with a different inbred variety toproduce hybrid seed. Generally, strategies that incorporate dominantsterility as a means to invoke male sterility cannot self-pollinate.This example provides such a method.

In some embodiments, when promoter inverted repeat strategies are usedto silence genes involved in male fertility (Loss of Function:LOF-plRmf), supplying an exogenous copy of the silenced gene restoresmale fertility. This is an example of restoration of fertility by Gainof Function (GOF-MF) (FIG. 4). As described previously, when silencingthe wheat MS45 gene and restoring using an exogenous source of thesuppressed fertility gene, the female inbreds are examples of LOF-plRmf,while the male restorers are examples of GOF-MF (FIG. 5).

It would be advantageous to generate an inbred maintainer population, toincrease the male sterile inbred line. To accomplish this, in oneembodiment for wheat, the maize MS45 promoter expressing the rice MS45gene (GOF-MF) is linked to the maize alpha amylase gene under control ofthe maize PG47 promoter and linked to a DsRed2 gene under control of thebarley LTP2 promoter (see, e.g., U.S. Pat. No. 5,525,716) and alsocarrying a PINII terminator sequence (GOF-MF-AA-DsRED). This constructis transformed directly into wheat by Agrobacterium-mediatedtransformation. Wheat plants containing single-copy GOF-MF-AA-DsREDcassette are emasculated and stigmas are fertilized with pollen frommale fertile plants containing LOF-plRmf and GOF-MF constructs. Seedsare harvested, screening by PCR for plants or seeds containing only theGOF-MF-AA-DsRED and LOF-plRmf TDNA insertions. These seeds are plantedand plants are allowed to self-pollinate. Red fluorescing seed fromthese selfed plants are planted and progeny screened by QPCR forhomozygous LOF-plRmf TDNA insertions. Seed from this generation ofprogeny segregates at a frequency of 1:1 red and non-red fluorescing.Red-fluorescing seed is hemizygous for GOF-MF-AA-DsRED, homozygous forLOF-plRmf, while non-fluorescing seed is homozygous for LOF-plRmf.Progeny of the non-fluorescing seed are male sterile and can be used asfemale inbreds during hybrid production. The red-fluorescing seedproduce progeny (hemizygous for GOF-MF-AA-DsRED; homozygous LOF-plRmf)that can be used to maintain and propagate the male sterile inbred.

Example 6. E. coli DNA (Adenosine-N6-)-Methyltransferase (DAM)Expression Affects Plant Fertility in Wheat

This example demonstrates that the fertility or fertility potential ofwheat plants can be altered by expression of E. coli DNA(Adenosine-N6-)-Methyltransferase (DAM) when under the control of themaize anther promoter 5126.

In maize, anther-directed expression of the E. coli DAM gene resulted ina high frequency of male sterile plants due to disruption of normaltapetum function (Unger, et al., (2001) Trans Res 10:409-422). However,it was not known whether expression of DAM in a polyploid plant wouldresult in male sterility.

Nucleic acid molecules and methods for preparing a vector to express inwheat plants, PHP56791, are similar to those previously described(Unger, et al., (2001) Trans Res 10:409-422). DNA sequence of the DAMgene was modified for expression in plants (SEQ ID NO: 11). Theoptimized DAM gene was placed under the transcriptional control of themaize 5126 promoter (Unger, et al., (2001) Trans Res 10:409-422) togenerate the plant transformation vector PHP56791. (SEQ ID NO: 12)PHP56791 was introduced into wheat Fielder variety byAgrobacterium-mediated transformation methods similar to those describedor referenced elsewhere herein.

Plants were grown in the greenhouse and transgene copy-number wasdetermined by quantitative polymerase chain reaction (QPCR). Plants weregrown to maturity and male fertility phenotype was recorded. As shown inTable 4, of the 85 primary T0 wheat transformants, 73 plants were malesterile while 12 plants were male fertile. Microscopic examination ofanthers from several independent PHP56791 plants revealed that theseanthers lacked pollen in contrast to similarly staged anthers fromuntransformed Fielder plants. In addition, anthers were consistentlyone-third to one-half the size of fully-developed fertile anthers anddid not contain microspores beyond the early vacuolate stage ofdevelopment. The small size of the anthers and lack of pollen inPHP56791 male sterile plants were similar to the male sterilityphenotypes observed in maize plants transformed with anther-expressedDAM gene.

These results demonstrate that the plant optimized DAM gene expressedfrom the maize anther promoter in PHP56791 is capable of generating malesterile wheat plants.

TABLE 4 Frequency of male sterility in plants containing PHP56791 TOTALSINGLE OR MULTI- PHP56791 EVENTS LOW COPY COPY MALE STERILE 73 46 27MALE FERTILE 12 8 4 85

Example 7. Preparation of Wheat Male Sterility Restorer Lines andRestoration of Male Fertility to PHP56791 Containing Wheat Plants

This example demonstrates that male-sterile plants containing constructPHP56791 can be restored to male fertility when also containing apromoter silencing construct.

In maize, promoter silencing constructs effectively transcriptionallysilence both endogenous and transformed promoters in planta (Cigan, etal., (2005) Plant Journal 43:929-940). This example was designed to testwhether a promoter inverted repeat designed to silence the maize antherpromoter, 5126, was capable of directing similar male sterilityphenotypes in wheat. In addition, if fertility was not impacted by themaize 5126 promoter inverted repeat, the experiment would determinewhether this silencing cassette could suppress the anther expression ofthe DAM gene in PHP56791 transgenic wheat plants.

Nucleic acid molecules and methods for preparing the plant vectorPHP54783 capable of suppressing the maize 5126 promoter used to expressthe DAM gene in PHP56791 are essentially as described for PHP20089(Cigan, et al., (2005) Plant Journal 43:929-940). PHP54783 (SEQ ID NO:13) was introduced into wheat Fielder variety by Agrobacterium-mediatedtransformation methods similar to those described or referencedelsewhere herein. Transformed plants were regenerated from tissueculture and grown in the greenhouse. Transgene copy-number wasdetermined by quantitative polymerase chain reaction (QPCR). Plants weregrown to maturity and male fertility phenotype was recorded.

All plants containing only the PHP54783 TDNA insertions were malefertile, suggesting that unlike expression of this pIR suppressioncassette in maize, the Zm5126 pIR does not result in male sterile wheatplants.

To determine whether the Zm5126 pIR silencing cassette was capable ofreversing the male sterility phenotype associated with PHP56791, pollenfrom two non-identical single-copy PHP54783 TDNA insertions (Male 1 andMale 2) were used to fertilize three non-identical, male sterile,PHP56791 plants (Female 1, 3, 4). Seed was harvested from these crosses,planted and progeny genotyped for the presence of PHP54783 and PHP56791TDNA insertions by PCR. Plants containing only PHP56791, or bothPHP56791 and PHP54783, were grown to maturity and male fertilityphenotype recorded. As shown in Table 5, Group 1 and 4 wheat plantscontaining only PHP56791 did not contain pollen and were male sterile(No Seed).

TABLE 5 Male Fertility phenotype of transgenic wheat plants containingDominant sterility construct PHP56791 and Restorer PHP54783. DominantSterility Construct RESTORER PLANT GROUP PHP56791 PHP54783 FEMALE MALESEED SET 1 1 + + 1 1 SEED 2 1 + 1 1 NO SEED 3 1 + 1 1 NO SEED 4 1 + 1 1NO SEED 1 3 + + 3 1 SEED 1 4 + + 4 2 SEED 2 4 + + 4 2 SEED 3 4 + + 4 2SEED 4 4 + 4 2 NO SEED 5 4 + 4 2 NO SEED 6 4 + 4 2 NO SEED

In contrast, PHP56791 plants also containing PHP54783 from Groups 1, 3and 4 shed pollen and were capable of self-fertilization (Seed). Seednumber per plant in PHP56791/PHP54783 progeny was similar to seednumbers obtained from untransformed Fielder variety plants. Theseresults demonstrate that the Zea mays 5126 promoter inverted repeat wascapable of restoring fertility to wheat plants containing the Dominantmale sterility construct PHP56791.

Example 8. Sources of Promoters and Gene Products to Confer MaleSterility and Restore Fertility in Wheat

The promoter expressing the E. coli DAM gene in PHP56791 can be ananther-preferred promoter such as the promoter of the maize MS45, BS7 orMS26 gene, or for example, the promoter of the rice or Arabidopsishomolog of the maize MS45, 5126, BS7 or MS26 gene, such that expressionby this plant promoter:DAM transcription unit renders wheat plants malesterile. In certain respects, it is advantageous to use non-wheatpromoters to express the DAM gene. For example, where promoter invertedrepeats from the same species have the potential to reduce targetpromoter function such that the plant is non-viable or non-reproductive,a promoter from a different species can be used to transcriptionallyexpress the dominant sterility gene (e.g., DAM), thus circumventing thispotential problem.

In addition, the E. coli DAM gene in PHP56791 can be replaced by sourcesother than DAM, for example barnase or another gene product that rendersplants male sterile as a result of reduced tapetum function or otherdisruption of development of male reproductive tissue.

Taken together, the present Examples demonstrate that a Dominant malesterility gene can be inactivated using pIR-mediated suppression andthat a fertile phenotype can be restored in genotypically sterileplants.

Example 9. Inbred Maintenance and Increase of LOF-DomMS Male SterilePlants Using a Hemizygous Maintainer

It would be advantageous to produce a pure line of male sterile plantsto allow for cross pollination with a different inbred variety toproduce hybrid seed. Generally, sterility strategies that includedominant approaches prevent plants from self-pollinating. This exampleprovides such a method.

In some embodiments, dominant male sterility is accomplished by theintroduction of a construct comprising a promoter driving a gene toexpress a gene product, such as a protein or RNA, that causes malesterile plants due to general or specific disruption of reproductivedevelopment, such as anther development, tapetum development ormicrospore function. In these Dominant Loss of Function (LOF-DomMS)examples, restoration of fertility could be accomplished byco-expressing an exogenous promoter inverted repeat (pIR) construct thatsilences the promoter (MSp) used to drive the Dominant sterility gene(MSpMS). This is an example of restoration of fertility by Gain ofFunction by promoter inverted repeats (GOF-pIRMSp) (FIG. 6). Asdescribed previously, disrupting normal tapetum function by Zm5126:DAM(MSpMS) is an example of the LOF-DomMS female inbred; restoration offertility using an exogenous source of the Zm5126pIR (pIRMSp) is anexample of GOF-pIRMSp (FIG. 7).

It would be advantageous to generate an inbred maintainer populationwhich could be used to increase the male sterile inbred line containingMSpMS. To accomplish this, the GOF-pIRMSp is linked to the maize alphaamylase gene under control of the PG47 promoter and linked to a DsRed2gene under control of the barley LTP2 promoter (see, e.g., U.S. Pat. No.5,525,716) and also carrying a PINII terminator sequence(GOF-pIRMSp-AA-DsRED). This construct is transformed directly into wheatby Agrobacterium-mediated transformation. Wheat plants containingsingle-copy GOF-pIRMSp-AA-DsRED cassette are emasculated and stigmas arefertilized with pollen from male fertile plants containingMSpMS/GOF-pIRMSp. Seeds are harvested, screening by PCR for plants orseeds containing only the GOF-pIRMSp-AA-DsRED and MSpMS TDNA insertions.Plants are allowed to self-pollinate. Red fluorescing seed from theseselfed plants are planted and progeny screened by QPCR for homozygousMSpMS TDNA insertions. Seed from this generation of progeny willsegregate at a frequency of 1:1 red and non-red fluorescing. Redfluorescing seed is hemizygous for GOF-pIRMSp-AA-DsRED and homozygousfor MSpMS; while non-fluorescing seed is homozygous for MSpMS. Progenyof the non-fluorescing seed are male sterile and can be used as femaleinbreds during hybrid production. The red fluorescing seed produceprogeny (hemizygous for GOF-pIRMSp-AA-DsRED; homozygous for MSpMS) thatwould be used to propagate the male sterile inbred. In the exampleabove, the MSpMS could be Zm5126DAM, while GOF-pIRMSp would correspondto Zm5126pIR.

Example 10. Embodiments

-   1. A breeding pair of plants, comprising: a first plant and a second    plant, wherein the first plant comprises an exogenous nucleic acid    molecule that when expressed suppresses the first plant's GOF-MF    gene (gain of function male fertility gene) or the first plant's    promoter driving the GOF-MF gene so that the first plant is male    sterile, and (b) a second plant, wherein the second plant expresses    a GOF-MF (gain of function male fertility) gene so that the second    plant is male-fertile and wherein the GOF-MF gene in the second    plant is able to restore male fertility when the second plant is    crossed with the first plant and the resulting progeny are male    fertile.-   2. The pair of plants of embodiment 1, wherein the GOF-MF gene is    endogenous or exogenous to the second plant.-   3. The pair of plants of embodiment 1, wherein the GOF-MF gene is a    MS45, MS26, or MS22 fertility gene.-   4. The pair of plants of embodiment 1, wherein the GOF-MF gene is    from a monocot or dicot.-   5. The pair of plants of embodiment 1, wherein the GOF-MF gene is    from wheat, maize, rice, sorghum, barley, rye, Arabidopsis, soybean    or sunflower.-   6. The pair of plants of embodiment 1, wherein the exogenous nucleic    acid molecule encodes a protein or is an RNAi, microRNA, an    antisense or hairpin molecule element.-   7. The pair of plants of embodiment 6, wherein in the first plant    the hairpin molecule suppresses expression of the GOF-MF gene    selected from the group consisting of: wheat, maize, rice, sorghum,    barley, rye, Arabidopsis, soybean or sunflower MS45, MS26 or MS22    gene.-   8. The pair of plants of embodiment 1, wherein the exogenous nucleic    acid molecule is operably linked to a promoter.-   9. The pair of plants of embodiment 8, wherein the promoter is an    inducible promoter, a constitutive promoter, a tissue preferred    promoter, a temporally regulated promoter or an element thereof.-   10. The pair of plants of embodiment 8, wherein the promoter is a    ubiquitin promoter.-   11. The pair of plants of embodiment 8, wherein the promoter is a    male reproductive tissue-preferred promoter.-   12. The pair of plants of embodiment 1, wherein the GOF-MF gene    (gain of function male fertility gene) in plant 2 comprises a    suppression element capable of suppressing expression of the    exogenous nucleic acid molecule suppressing the GOF-MF gene or    promoter driving the GOF-MF gene in the first plant so that when    plant 1 and plant 2 are crossed the resulting progeny are    male-fertile.-   13. The pair of plants of embodiment 12, wherein the suppression    element in the second plant encodes a protein or is an RNAi,    microRNA, an antisense or hairpin molecule element that silences the    promoter driving the exogenous nucleic acid or the exogenous nucleic    acid nucleic acid, thereby restoring fertility to the resulting    progeny.-   14. The pair of plants of embodiment 12, wherein the suppression    element in the second plant is a hairpin that targets the promoter    driving the expression of the exogenous nucleic acid molecule, such    that the resulting progeny is male-fertile.-   15. The pair of plants of embodiment 1, wherein the GOF-MF gene in    the second plant is a GOF-MF gene that is the same as the GOF-MF    gene being suppressed in the first plant.-   16. The pair of plants of embodiment 1, wherein the sequence    encoding the GOF-MF gene in the second plant is a GOF-MF gene that    is suppressed in the first plant, is different than the GOF-MF gene    in the first plant but is capable of restoring fertility to progeny    resulting from crossing plant 1 with plant 2.-   17. A method of restoring male fertility in a plant exhibiting male    sterility, comprising crossing the breeding pair of plants of    embodiment 1.-   18. A method of restoring male fertility in a plant exhibiting male    sterility, comprising crossing the breeding pair of plants of    embodiment 6.-   19. A plant produced by the method of embodiments 17 or 18, wherein    said plant comprises an exogenous nucleic acid molecule that when    expressed, suppresses the plant's GOF-MF gene (gain of function male    fertility gene) or the plant's promoter driving the GOF-MF gene and    a second plant, wherein the second plant expresses a GOF-MF (gain of    function male fertility) gene so that the plant is male-fertile.-   20. Cells of the plant of embodiment 19.-   21. Seed or progeny of the plant of embodiment 19.-   22. Seed or progeny of the plant of embodiment 19 comprising the    GOF-MF gene and the exogenous nucleic acid construct.-   23. A method of generating a male-fertile plant, comprising crossing    the breeding pair of any of the embodiments of 1-16.-   24. A plant produced by the method of embodiment 23.-   25. A cell of the plant of embodiment 24.-   26. Seed or progeny of the plant of embodiment 24.-   27. A method of increasing male sterile seeds comprising:    -   (a) emasculating a first plant that is hemizygous for the GOF-MF        (gain of function male fertility) gene and wherein the GOF-MF        (gain of function male fertility) gene is operably linked to a        gene that disrupts the function and/or development of        male-reproductive tissue and a marker gene;    -   (b) pollinating the first plant with pollen from a second plant,        wherein the plant is male fertile and comprising an exogenous        nucleic acid construct that targets the second plant's GOF-MF        gene, and wherein the second plant comprises a GOF-MF gene (gain        of function male fertility gene) that maintains fertility in the        second plant;    -   (c) harvesting seeds;    -   (d) selecting seeds comprising the GOF-MF (gain of function male        fertility) gene operably linked to the gene that disrupts the        function and/or development of male-reproductive tissue and the        marker gene and the exogenous nucleic acid construct that        targets the second plant's GOF-MF gene or promoter;    -   (e) planting the seeds from step (d);    -   (f) allowing the plants grown from the seeds of step (d) to        self-pollinate;    -   (g) harvesting seeds from the resulting progeny of step (f);    -   (h) selecting the seeds harvested in step (g) for seeds that        express the marker, wherein the seeds that express the marker        are hemizygous for the GOF-MF gene linked to the gene that        disrupts the function and/or development of male-reproductive        tissue and the marker gene and homozygous for the exogenous        nucleic acid;    -   (i) planting the seeds from step (h);    -   (j) allowing the plants grown from the seeds of step (i) to        self-pollinate;    -   (k) harvesting seeds from the resulting progeny of step (j);    -   (l) selecting seeds harvested in step (k) for seeds that are        homozygous for the exogenous nucleic acid construct that targets        the plant's GOF-MF gene and do not contain the marker gene.-   28. A method of increasing male-sterile plants comprising:    -   a. pollinating plants grown from the seeds of step (l) of        embodiment 27 with pollen from the plant comprising the GOF-MF        (gain of function male fertility) gene operably linked to the        gene that disrupts the function and/or development of        male-reproductive tissue and the marker gene and the exogenous        nucleic acid construct that targets the second plant's GOF-gene;        and    -   b. harvesting the seeds from the resulting progeny of step (a)        wherein the seeds that homozygous for the exogenous nucleic acid        suppression molecule are male-sterile and do not contain the        marker gene.-   29. The method of embodiment 28, wherein the gene that disrupts the    function and/or development of male-reproductive tissue is alpha    amylase or barnase.-   30. The method of embodiment 28, wherein the marker gene is a    selectable or scorable marker.-   31. A method of producing male-sterile female plants, the method    comprising:    -   a. planting the seeds from step (l) from the embodiment of claim        27; and    -   b. allowing the seeds to grow into plants, wherein the resulting        plants are homozygous for the exogenous nucleic acid construct        and male-sterile.-   32. A method of producing hybrid plant seed, comprising:    -   pollinating plants homozygous for the nucleic acid molecule that        suppresses the GOF-MF gene or the promoter driving the GOF-MF        gene with a male fertile plant.-   33. A method of producing hybrid plant seed, comprising:    -   pollinating the male-sterile female plants of embodiment 31 with        a male fertile plant.-   34. Hybrid seed produced by the method of embodiments 32 or 33.-   35. A method of obtaining a hybrid plant, comprising growing the    hybrid seed of embodiment 34.-   36. A hybrid plant produced by the method of embodiment 35.-   37. A breeding pair of plants, comprising:    -   a first transgenic plant and a second transgenic plant, wherein        the first transgenic plant is homozygous for an expressible        exogenous nucleic acid molecule that when expressed suppresses        the first plant's GOF-MF gene or the promoter driving the first        plant's GOF-MF gene so that the plant is male-sterile; and        wherein the second transgenic plant is hemizygous for the GOF-MF        gene operably linked to a gene that disrupts the function and/or        development of male-reproductive tissue and operably linked to a        marker and homozygous for an exogenous nucleic acid construct        that targets the second plant's GOF-MF gene, that maintains        fertility in the second plant.-   38. The pair of plants of embodiment 37 wherein the sequence    encoding the GOF-MF gene in the second plant is a homolog of the    GOF-MF gene that is suppressed in the first plant.-   39. The pair of plants of embodiment 36, wherein the GOF-MF gene is    from a monocot or dicot.-   40. The pair of plants of embodiment 36, wherein the restorer gene    is from wheat, maize, rice, sorghum, barley, rye, soybean,    Arabidopsis or sunflower.-   41. The pair of plants of embodiment 36 wherein the sequence    encoding the GOF-MF gene in the second plant is operably linked to a    promoter.-   42. The pair of plants of embodiment 41, wherein the promoter    driving the GOF-MF gene is from a different species than the species    of the promoter driving expression of the GOF-MF gene.-   43. The pair of plants of embodiment 41, wherein the promoter in the    second plant is a MS45, MS26, 5126, BS7 or MS22 gene promoter.-   44. The embodiments of 1-43, wherein the pair of plants are    polyploid.-   45. The embodiments of 1-44, wherein the pair of plants are    hexaploid.-   46. A method of maintaining a male-sterile plant comprising:    -   a. crossing the pair of plants in embodiments 37-45.

Example 11. Embodiments—LOF

-   1. A breeding pair of plants, comprising: a first plant and a second    plant, wherein the first plant expresses a dominant male sterility    gene so that the first plant is male-sterile, and (b) a second    plant, wherein the second plant comprises an expressible exogenous    nucleic acid molecule comprising a polynucleotide that when    expressed suppresses the expression of the dominant male sterility    gene or the promoter operably linked to the dominant male sterility    gene of the first plant.-   2. The pair of plants of embodiment 1, wherein expression of the    dominant male sterility gene is DAM, streptavidin, MS44 mutant, or    barnase.-   3. The pair of plants of embodiment 1, wherein expression of the    dominant male sterility gene is plant codon optimized.-   4. The pair of plants of embodiment 1, wherein, in the first plant,    the dominant male sterility gene is operably linked to a promoter.-   5. The pair of plants of embodiment 4, wherein the promoter is an    inducible promoter, a constitutive promoter, a tissue preferred    promoter, a temporally regulated promoter or an element thereof.-   6. The pair of plants of embodiment 4 wherein the promoter is an    anther-specific promoter.-   7. The pair of plants of embodiment 4, wherein the anther-specific    promoter is MS45, MS26, MS22, or 5126 gene promoter.-   8. The pair of plants of embodiment 4 wherein the promoter is a    tassel-preferred promoter.-   9. The pair of plants of embodiment 1, wherein the exogenous nucleic    acid molecule in the second plant encodes a protein or is an RNAi,    an antisense or hairpin suppression element.-   10. The pair of plants of embodiment 1, wherein the polynucleotide    in the second plant that suppresses the expression of the exogenous    nucleic acid molecule in the first plant is operably linked to a    promoter.-   11. The pair of plants of embodiment 10, wherein the promoter is an    inducible promoter, a constitutive promoter, a tissue preferred    promoter, a temporally regulated promoter or an element thereof.-   12. The pair of plants of embodiment 10, wherein the promoter is an    anther-specific promoter.-   13. The pair of plants of embodiment 10, wherein the promoter is an    anther-specific promoter is the MS45, MS26, MS22, or 5126 gene    promoter.-   14. The pair of plants of embodiment 10, wherein the promoter is a    tassel-preferred promoter-   15. The pair of plants of embodiment 1, wherein the first and second    plants are polyploid.-   16. The pair of plants of embodiment 1, wherein the first and second    plants are monocots or dicots.-   17. The pair of plants of embodiment 1, wherein the first and second    plants are wheat, maize, rice, sorghum, barley, rye, Arabidopsis,    soybean or sunflower.-   18. A method of restoring male fertility in a plant exhibiting male    sterility, comprising crossing the breeding pair of plants of    embodiments 1-17.-   19. A plant produced by the method of embodiment 18, wherein said    plant is male-fertile and is hemizygous for the dominant male    sterility gene and the exogenous nucleic acid molecule.-   20. Cells of the plant of embodiment 19.-   21. Seed or progeny of the plant of embodiment 19.-   22. A method of increasing male-sterile female seeds comprising:    -   (a) emasculating a first plant, wherein the first plant is        hemizygous for an expressible exogenous nucleic acid comprising        a polynucleotide that when expressed suppresses the expression        of a dominant male sterility gene or a promoter operably linked        to the dominant male sterility gene so that the first plant is a        male-sterile female plant, and wherein the expressible exogenous        nucleic acid is operably linked to a gene that disrupts the        function and/or development of male-reproductive tissue and a        marker gene;    -   (b) pollinating the first plant with pollen from male-fertile        plants hemizygous for the expressible exogenous nucleic acid        molecule comprising the polynucleotide that when expressed,        suppresses the expression of the dominant male sterility gene in        plant 1 or the promoter operably linked to the dominant male        sterility gene in plant 1 and hemizygous for a dominant        sterility gene;    -   (c) harvesting seeds;    -   (d) selecting seeds that are hemizygous for the expressible        exogenous nucleic acid linked to the gene that disrupts the        function and/or development of male-reproductive tissue and the        marker gene and hemizygous for the dominant male sterility gene;    -   (e) planting the seeds from step (d);    -   allowing the plants grown from the seeds of step (d) to        self-pollinate; and    -   (g) harvesting seeds from step (f);    -   (h) selecting seeds that are hemizygous for the expressible        exogenous nucleic acid linked to the gene that disrupts the        function and/or development of male-reproductive tissue and the        marker gene and homozygous for the dominant sterility gene;    -   (i) planting the seeds from step (h);    -   (j) allowing the plants grown from the seeds of step (h) to        self-pollinate; and    -   (k) harvesting seeds from the plants in step (j); and    -   (l) selecting seeds that are homozygous for the dominant        sterility gene;-   23. A method of producing male-sterile female plants, the method    comprising:    -   a. planting the seeds from step (l) of embodiment 22; and    -   b. allowing the seeds to grow into plants, wherein the resulting        plants are homozygous for the dominant sterility gene.-   24. A method of producing hybrid plant seed, comprising:    -   pollinating the male-sterile female plants homozygous for the        dominant sterility gene of embodiment 23 with a male-fertile        inbred plant.-   25. The method of embodiment 23, wherein the dominant male sterility    gene is DAM, streptavidin, MS44 mutant or barnase.-   26. The method of embodiment 23, wherein expression of the dominant    male sterility gene is plant codon optimized.-   27. The method of embodiment 23, wherein the dominant male sterility    gene is operably linked to a promoter.-   28. The method of embodiment 27, wherein the promoter is an    inducible promoter, a constitutive promoter, a tissue preferred    promoter, a temporally regulated promoter or an element thereof.-   29. The method of embodiment 27, wherein the promoter is an    anther-specific promoter.-   30. The method of embodiment 29, wherein the anther-specific    promoter is MS45, MS26, MS22, or 5126 gene promoter.-   31. The method of embodiment 29, wherein the promoter is a    tassel-preferred promoter.-   32. The method of embodiment 23, wherein the plants are polyploid.-   33. The method of embodiment 23, wherein the plants are monocots or    dicots.-   34. The method of embodiment 23, wherein the plants are wheat, corn,    maize, rice, sorghum, barley, rye, soybean, Arabidopsis or    sunflower.-   35. Hybrid seed produced by the method of embodiments 24-34.-   36. A method of obtaining a hybrid plant, comprising growing the    hybrid seed of embodiment 35.-   37. The method of embodiment 22, wherein the gene that disrupts the    function and/or development of male-reproductive tissue gene is    alpha amylase or barnase.-   38. The method of embodiment 22, wherein the marker gene is a    selectable or scorable marker.-   39. A hybrid plant produced by the method of embodiment 36.-   40. A method of propagating a male-sterile plant comprising:    -   a. crossing a first plant that is hemizygous for an expressible        exogenous nucleic acid comprising a polynucleotide that when        expressed suppresses the expression of a dominant male sterility        gene or a promoter operably linked to the dominant male        sterility gene so that the first plant is a male-sterile female        plant, and wherein the expressible exogenous nucleic acid is        operably linked to a gene that disrupts the function and/or        development of male-reproductive tissue and a marker gene with a        plant from the embodiment of 23.-   41. A male-sterile female plant produced by the method of embodiment    40.

That which is claimed:
 1. A method of modulating fertility in apolyploid plant, comprising expressing in said plant a constructtargeting a promoter, wherein said promoter drives expression of a geneproducing a gene product which influences fertility.
 2. The method ofclaim 1 wherein the construct comprises polynucleotides comprisingsubstantial identity to the target promoter, wherein saidpolynucleotides are provided in an inverted repeat orientation.
 3. Themethod of claim 2 wherein the construct comprises a consensus sequencederived from the MS45 promoter sequences of two or more of the A, B, andD genomes of wheat.
 4. A construct effective for modulating fertility ina polyploid plant, said construct comprising polynucleotides comprisingsubstantial identity to a promoter which drives expression of a geneproducing a gene product which influences fertility, wherein saidpolynucleotides are provided in an inverted repeat orientation.
 5. Amethod of restoring fertility to a polyploid plant which comprises afirst construct of claim 4, comprising introducing into said plant asecond construct comprising a promoter operably linked to apolynucleotide, wherein said polynucleotide encodes a gene product,expression of which gene product complements the modulation of fertilityresulting from the first construct.
 6. The method of claim 6, whereinsaid introducing is by transformation or by crossing.
 7. An isolatednucleic acid molecule comprising a polynucleotide selected from thegroup consisting of: (a) a nucleotide sequence comprising the nucleotidesequence of SEQ ID NO: 1, 2 or 3; (b) a nucleotide sequence comprising afragment or variant of the nucleotide sequence of SEQ ID NO: 1, 2 or 3,wherein the sequence initiates transcription in a plant cell; (c) apolynucleotide which is complementary to the polynucleotide of (a) or(b).
 8. An expression cassette comprising the polynucleotide of claim 15operably linked to a heterologous polynucleotide of interest.
 9. A plantcomprising the expression cassette of claim
 8. 10. A method forexpressing a polynucleotide in a plant or a plant cell, said methodcomprising introducing into the plant or the plant cell an expressioncassette comprising a promoter operably linked to a heterologouspolynucleotide of interest, wherein said promoter comprises a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence comprising the nucleotide sequence of SEQ ID NO: 1, 2 or 3; (b)a nucleotide sequence comprising a fragment or variant of the nucleotidesequence of SEQ ID NO: 1, 2 or 3, wherein the sequence initiatestranscription in a plant cell; (c) a nucleotide sequence which iscomplementary to (a) or (b).
 11. The method of claim 10, wherein saidheterologous polynucleotide of interest is expressed preferentially inreproductive tissue of a plant.
 12. A construct effective for inhibitingfertility in a polyploid plant, the construct comprising a promoterfunctional in plants, operably linked to a polynucleotide encoding agene product which influences fertility.
 13. The construct of claim 12wherein the gene product is DAM, optimized DAM, MS44, streptavidin orbarnase.
 14. A breeding pair of plants, comprising: a first plant and asecond plant, wherein the first plant expresses an exogenous dominantmale sterility gene so that the first plant is male-sterile, and (b) asecond plant, wherein the second plant comprises an expressibleexogenous nucleic acid molecule comprising a polynucleotide that whenexpressed suppresses the expression of the dominant male sterility geneor the promoter operably linked to the dominant male sterility gene ofthe first plant.
 15. The breeding pair of plants of claim 14, whereinthe dominant male sterility gene impacts anther development, tapetumdevelopment, or microspore function.
 16. The breeding pair of plants ofclaim 14, wherein the dominant male sterility gene is selected from thelist consisting of streptavidin, DAM, and barnase.
 17. The breeding pairof plants of claim 14, wherein the expressible exogenous nucleic acidmolecule of the second plant comprises a promoter inverted repeat (pIR)targeting the promoter operably linked to the dominant male sterilitygene of the first plant.
 18. A breeding pair of plants, comprising afirst plant and a second plant, wherein the first plant expresses a pIR(promoter inverted repeat) directed to a first promoter operably linkedto a fertility gene, wherein said first promoter and gene may beheterologous or natively linked with respect to each other, and whereinexpression of said pIR results in male sterility of the first plant; andwherein the second plant comprises a construct comprising apolynucleotide, expression of which will complement the male-sterilityof the first plant, and wherein the polynucleotide is operably linked toa second promoter which would not be impacted by the pIR of the firstplant; wherein crossing the plants restores fertility to progeny of thefirst plant.
 19. The breeding pair of plants of claim 18, wherein thepIR is operably linked to a promoter which expresses preferentially inmale reproductive tissue.
 20. The method of claim 1 wherein the targetedpromoter is endogenous.